A ww homogametic male decapod crustacean and methods of using the same

ABSTRACT

The present invention provides an all-female progeny of a decapod crustacean. The invention further provides the newly created: WW homogametic Neo-male decapod crustacean and WW homogametic female decapod crustacean. The crossing of WW homogametic Neo-male decapod crustacean and WW homogametic female decapod crustacean resulted in a 100% homogametic all-WW female progeny.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. PatentApplication No. 62/254,264, filed Nov. 12, 2015, entitled “HOMOGAMETICFEMALE DECAPOD CRUSTACEAN AND METHODS OF USING THE SAME”, the contentsof which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

This invention is directed to; inter alia, a homogametic WW male decapodcrustacean and to methods of obtaining an all-female progeny by crossinga homogametic WW decapod crustacean with another decapod crustacean.

BACKGROUND OF THE INVENTION

The attributes of gender selection in animal breeding and the ability toform an agro-technical infrastructure around it which will improveperformance is already well established in cultured animals such ascattle, poultry and fish (De Vries (2008); Correa et al., (2005) andBeardmore et al., (2001)). Crustaceans' aquaculture still predominantlyuses heterogenic populations which hinders full utilization of valublesexual dimorphic growth patterns. When segregated based on gender,crustaceans demonstrate enhanced growth rates as energy resources areallocated mainly towards somatic growth rather than gonad maturation andother reproduction-related activities. Predominantly, the crustaceanaquaculture depends on nurseries & hatcheries for juvenile supply, asthe vast majority of the industry is comprised of grow-out farms whichseek to prevent undesirable reproduction and allocation of energytowards it. Thus, culturing of mono-sex populations, either all-male orall-female, produces higher yields and greater commercial value.Although in some crustacean species all-male populations generate higheryields under extensive culturing conditions, several studies suggest(Gopal et al., (2010) and Otoshi et al., (2003)) that under intensifiedfarming conditions and in most decapod crustacean species farmed todayit is females that are economically more favorable, as they grow largerand suggested to have better feed conversion than males (Moss and Moss(2006)). In an all-female population under commercial conditions, finalsizes at harvest are highly uniform yielding up to 35% higher productionvalue than all-males. Furthermore, it appears that the separation fromthe males reduces aggressiveness and stress, decreases cannibalism,delivers a higher homogeneity in marketing size and most importantlyenables higher stocking rates, all of which are highly valued bygrowers. On the same note, the supplementation of only WW females tohatcheries, which will provide the all-WZ progenies to the growers, willtransform the traditional culturing of crustaceans into a ‘seed-like’sustainable industry. As F2 (or later generations) inbreeding whichproduces inferior seedstocks will not take place at local hatcheries,breeding companies will be able to protect their proprietary elite lines(which have been highly selected for over tens of generations.

Sexual differentiation and the development of secondary sexualcharacteristics are controlled by different mechanisms across evolution.In vertebrates and some invertebrate groups, these processes are underthe control of sex hormones. Given the confirmation that insectsprobably have no sex hormones, the agents responsible for the sexualmaturation of arthropods remain under debate. Crustaceans that areevolutionary close to insects possess an androgenic gland (AG) which isresponsible for male sexual differentiation. Interestingly, in somecrustaceans, endocrine regulation of sexual differentiation precedes thephenotypic appearance of this secondary external feature.

The role of the AG in male sexual differentiation was demonstrated inseveral crustacean species by observing primary and secondary sexcharacteristics after AG removal or transplantation. In the amphipodOrchestia gamarella, bilateral AG ablation diminished spermatogenesisand obstructed the development of secondary male characteristics. InMacrobrachium rosenbergii, a fully functional sex reversal from males toNeo-females and from females to Neo-males was achieved by surgicalbilateral AG ablation and transplantation, respectively, with the lattershowing poor efficacy thus failed to reach commercial materialization.

Contrast to the above, a revolutionized technology has recently shown tosuccessfully transform females of the prawn M. rosenbergii into fullyfunctional and reproductive Neo-males. This technology relies on asingle injection of primary AG cell suspension (Levy et al., (2016)).While the technology is highly efficient, cost-effective and safe forproduction of mono-sex all-female populations of decapod crustaceans,the produced WW females (25% of each progeny) are isolated based ongenotyping. To date, a breeding counterpart male for incrossing with aWW homogametic female while preserving the trait-of-interest in 100% ofthe progeny (i.e. homogametic WW), is yet to be produced.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a homogametic WWdecapod crustacean.

In another embodiment, the present invention further provides a male ora Neo-male homogametic WW decapod crustacean.

In another embodiment, the present invention further provides a methodfor obtaining an all-female progeny, comprising the step of mating a WWdecapod crustacean with another decapod crustacean. In anotherembodiment, the present invention further provides a method forobtaining an all-homogoametic WW female progeny.

In another embodiment, the present invention further provides a methodfor obtaining an all-female progeny, comprising the step of mating a WWfemale decapod crustacean with a WZ Neo-male decapod crustacean.

In another embodiment, the present invention further provides a methodfor obtaining an all-female progeny, comprising the step of crossing aWW Neo-male decapod crustacean with a WW female decapod crustacean. Inanother embodiment, the present invention further provides a water tankcomprising a WW Neo-male and a WW female.

In another embodiment, the present invention further provides a methodfor obtaining a WW Neo-male decapod crustacean, comprising the step ofinjecting to a decapod crustacean comprising the WW sex chromosomes andyounger than 180 days post-larva, a composition comprising at least1×10² solitary cells derived from an androgenic gland (AG) of a decapodcrustacean, thereby obtaining a WW Neo-male decapod crustacean. Inanother embodiment, an androgenic gland (AG) is a hypertrophiedandrogenic gland.

In another embodiment, the present invention further provides a femaledecapod crustacean obtained by any of the present methods. In anotherembodiment, the present invention further provides a WW female decapodcrustacean obtained by any of the present methods.

In another embodiment, the present invention further provides anall-female progeny obtained by any of the present methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the two-phase procedure for massproduction of all-female populations. ♀—represents females and♂—represents males. The letters W and Z are used to illustrate thechromosome-based sex determination model. WZ and WW are either femalesor Neo-males (i.e., sex-reversed females) and ZZ are males. × describesbreeding and arrows point to the progeny of each crossing. Percentages(%) reflect ratios in each progeny comparing males vs. females ornatural females (WZ) vs. homogametic females (WW). Seringe icon refersto the administration of enzymatically-dissociated AG cell suspensionculture. Phasel refers to the production of a WZ Neo-male and crossingit with a WZ female to obtain 25% WW females. Thereafter, WW females arecrossed with ZZ natural males to obtain all-WZ female populations. Phase2 refers to the manipulation of WW females to produce WW Neo-males andincrossing of the latter with WW females for mass production of 100%all-WW female populations.

FIG. 2: A micrograph of a histological comparison of intact andhypertrophied androgenic glands of M. rosenbergii. (A) Sections of anintact androgenic gland (AG) and (B) a hypertrophied AG which werestained using hematoxylin and eosin and observed under light microscope.The location of the AG is pointed out (arrows). Sperm Duct (SD). Scalebar represents 50 μm.

FIG. 3: A micrograph of an enzymatically-dissociated hypertrophied AGprimary cell culture in suspension from M. rosenbergii across time. AGcells were documented (A) 2 days and (B) 6 days after being seeded usinginverted light microscope. The location of the AG cells in the cultureis pointed out (arrow). Scale bar=500 μm (A) and 100 μm (B).

FIG. 4: Micrographs showing injection of enzymatically-dissociatedhypertrophied primary AG cell culture in suspension using amicro-injector apparatus to post larvae M. rosenbergii females. (A) Apost larvae is first restrained on a plasticine surface before (B) beingplaced under a laboratory binocular for the operation of amicro-injector. (C) The insertion of a glass capillary (highlighted bycircle) of the micro-injector (top side of the image) in the injectionprocedure of the cell suspension.

FIG. 5: Micrographs showing external secondary sex characters in M.rosenbergii. (A) Image of a male (♂) and two females (♀)—source: EudesCorreia. (B) Dissecting microscope images of the 2^(nd) pleopod of amale (top image) and female (bottom image) M. rosenbergii. The masculinepleopod contains both appendix masculina (white arrow) and appendixinterna (black arrow) while the feminine pleopod contains only appendixinterna.

FIG. 6: A gel micrograph showing genetic sex determination of WZNeo-male PLs. To discriminate the morphologically-indistinguishablemales from Neo-males, all the representative PLs which were manipulatedby injection and examined for the development (✓) or lack of (x)appendix masculina (AM) and masculine gonopores, were geneticallyexamined using a female-specific DNA sequence. The latter was amplifiedby means of PCR and was separated through a 2% agarose gel stained byEthidium Bromide and visualized using UV (upper panel). Mr β-actinserved as a positive control for PCR, ensuring the presence of DNA ineach sample (lower panel). DNA of a M. rosenbergii female (♀) and waterserved as a positive and negative control (NC), respectively.

FIG. 7: Micrographs showing M. rosenbergii WZ Neo-males develop hallmarkmale sex characteristics. Single injection of AG cell suspension inducesthe development of specific sex characteristics restricted to male M.rosenbergii: appendix masculina on the 2^(nd) pleopod (white arrow) andgonopores at the bases of the 5^(th) pereiopods (arrows).Morphologically, males (left column) and Neo-males (middle column) areindistinguishable from one another. Females bear only appendix interna(black arrow) and do not develop gonopores at the base of the 5^(th)pereiopods (right column).

FIG. 8: Micrographs showing M. rosenbergii WZ Neo-males achieve fullmasculine morphotypic differentiation. Neo-males resulting from a singleAG cell suspension progressed through normal morphotypic differentiationand developed into fully mature blue-claw, as determined according toconventional measuring system developed by Kuris et al., (1987). TheNeo-male's propodus (claw) grew to nearly twice as long as the carapace(70 mm compared with 37 mm, respectively). A large testicular tissue wasalso observed upon dissection (inset).

FIG. 9: Micrographs showing M. rosenbergii WZ Neo-male shows fullmasculine gonadogenesis. Histological cross sections stained Hematoxylinand Eosin reveal (A) a sperm-filled sperm duct along with (B) highlyactive testicular lobules. In the latter highly (dividing)—and lightly(mature)—dense regions were observed. (C) Sperm within the sperm ductshowed the characteristic inverted-umbrella morphology of a true Mrmature sperm. (D) Round large spermatogonium (Sg) cells are heavilystained and found to be located in the periphery of a lobule as opposeto spermatozoa (Sz) which were lightly stained and accommodate majorityof a lobule's volume, as is the case in sexually reproductive M.rosenbergii males. Bar=250 μm (A and B) and 50 μm (C and D). Fieldsdefined in frames in A and B were enlarged and documented as C and D,respectively.

FIG. 10: Molecular progeny test of WZ Neo-male crossed with a naturalfemale: A gel micrograph showing genetic sex determination of selectedPLs from the progeny of a Neo-male crossed with a natural female. Todiscriminate the morphologically-indistinguishable males from females(at the early age of PL₂₀), all the representatives were geneticallyexamined using a dual W- and Z-specific DNA sex markers. The latter wereamplified by means of multiplexed PCR and were separated through a 2%agarose gel stained by Ethidium Bromide and visualized using UV.

FIG. 11: Pie charts showing genotypic distribution of WZNeo-male×natural female progeny. The genotypes with respect to geneticsex of PLs from the progeny of a Neo-male and a natural female weredetermined using the molecular sex markers based on which finaldistributions were calculated. (A) The total distribution of 865 PLsbased on solely the Z-specific sex marker revealed that approximatelythree quarters of the progeny (77%) is comprised of WZ females and ZZmales. The remaining PLs (23%) are WW females. (B) A representativegroup of 174 PLs were sampled (from the 865 PLs) and geneticallyanalyzed using both the W- and Z-specific sex markers. The finalgenotypic sex determination indicated that WW females and ZZ males areat 1:1 ratio (24.1% each) and are both at approximately 1:2.1 ratio withnatural WZ females (51.8%).

FIG. 12: Molecular progeny test of WW female crossed with natural ZZmale give rise to all-WZ female population: A gel micrograph showinggenetic sex determination of selected PLs from an all-female progeny ofa WW female crossed with a natural ZZ male. All the representatives weregenetically examined using a dual W- and Z-specific DNA sex markers. Thelatter were amplified by means of multiplexed PCR and were separatedthrough a 2% agarose gel stained by Ethidium Bromide and visualizedusing UV.

FIG. 13: Micrographs showing M. rosenbergii WW Neo-males achieve fullmasculine morphotypic differentiation. Neo-males resulting from a singleAG cell suspension progressed through natural morphotypicdifferentiation from small males (SM)—orange claw (OC) to fully matureblue-claw (BC), as determined according to conventional measuring systemdeveloped by Kuris et al., (1987) (A). The representative WW Neo-maleswere genetically examined using a dual W- and Z-specific DNA sexmarkers. The latter were amplified by means of multiplexed PCR and wereseparated through a 2% agarose gel stained by Ethidium Bromide andvisualized using UV. Scale bar=5 cm (B).

FIG. 14: Micrographs showing M. rosenbergii WW Neo-males develophallmark male sex characteristics. Single injection of AG cellsuspension induces the development of specific sex characteristicsrestricted to male M. rosenbergii: appendix masculina on the 2^(nd)pleopod (white arrow) and gonopores at the bases of the 5^(th)pereiopods (arrows). Morphologically, males (left column) and Neo-males(middle column) are indistinguishable from one another. Females bearonly appendix interna (black arrow) and do not develop gonopores at thebase of the 5^(th) pereiopods (right column).

FIG. 15: Micrographs showing M. rosenbergii WW Neo-males achievemasculine development. Neo-males resulting from a single AG cellsuspension showed complete gonadogenesis resulting with a testiculartissue (A, enframed). Histological cross sections stained Hematoxylinand Eosin reveal (B) active testicular lobules. In the latter, bothdividing and mature regions were observed. (C) Round largespermatogonium (Sg) cells are heavily stained and found to be located inthe periphery of a lobule as oppose to spermatozoa (Sz) which werelightly stained and accommodate majority of a lobule's volume, as is thecase in sexually reproductive M. rosenbergii males. Bar=1 cm (A), 250 μm(B) and 50 μm (C). Field defined in frame in B was enlarged anddocumented as C.

FIG. 16: Molecular progeny test of WW Neo-male crossed with WW femalegive rise to all-WW female population: A gel micrograph showing geneticsex determination of selected PLs from an all-WW female progeny of a WWNeo-male crossed with a WW female. All the representatives weregenetically examined using a dual W- and Z-specific DNA sex markers. Thelatter were amplified by means of multiplexed PCR and were separatedthrough a 2% agarose gel stained by Ethidium Bromide and visualizedusing UV.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides a WW decapodcrustacean. In one embodiment, a decapod crustacean is a Penaeid shrimp.In one embodiment, a decapod crustacean is a fresh water prawn. In oneembodiment, a decapod crustacean is of the genus Macrobachium. Inanother embodiment, a decapod crustacean is Macrobrachium rosenbergii.

In another embodiment, homogametic WW decapod crustacean is a male. Inanother embodiment, homogametic WW decapod crustacean is a female. Inanother embodiment, homogametic WW decapod crustacean is a Neo-male.

In one embodiment, the present invention provides a method for obtainingan all-female progeny, comprising the step of mating a WW decapodcrustacean with another decapod crustacean. In another embodiment, anall-female progeny is a progeny that consists 100% WW females. Inanother embodiment, an all-female progeny is a progeny that consists100% WW females all of which are full sisters. In another embodiment, anall-female progeny is a progeny that consists 100% WW females all ofwhich have a common maternal parent. In another embodiment, anall-female progeny is a progeny that consists 100% WW females, all ofwhich have parents in which the paternal parent and maternal parent areWW. In another embodiment, an all female progeny is a progeny devoid ofa male. In another embodiment, an all female progeny is a progenycomprising at least 500 females. In another embodiment, an all femaleprogeny is a progeny comprising at least 1000 females. In anotherembodiment, an all female progeny is a progeny comprising at least 3000females. In another embodiment, an all female progeny is a progenycomprising at least 5000 females. In another embodiment, an all femaleprogeny is a progeny comprising at least 10000 females. In anotherembodiment, an all female progeny is a progeny comprising at least 25000females. In another embodiment, an all female progeny is a progenycomprising at least 50000 females. In another embodiment, an all femaleprogeny is a progeny comprising at least 100000 females. In anotherembodiment, an all female progeny is a progeny comprising at least150000 females. In another embodiment, a female comprises female sexchromosomes. In another embodiment, a female comprises ovaries andoviducts. In another embodiment, a female produces eggs.

In one embodiment, a method for obtaining an all-female progeny is amethod for obtaining a female decpod crustacean bearrying only femaleembryos. In one embodiment, “female embryos” are fertilized eggscomprising only female sex cromosomes. In one embodiment, “femaleembryos” consists fertilized eggs consisting female sex cromosomes.

In one embodiment, the invention provides a female decpod crustaceanbearrying only female embryos. In one embodiment, the invention providesa female decpod crustacean bearrying only embryos which consist femalesex cromosomes.

In one embodiment, the invention provides a water tank comprising a maledecapod crustacean and a female decapod crustacean, wherein the male isa WW Neo-male and the female is a WW female. In one embodiment, thewater tank is devoid of a male decapod crustacean or a female decapodcrustacean having sex chromosomes other than WW.

In one embodiment, the present invention provides a method for obtainingan all-female progeny, comprising the step of mating a female WW decapodcrustacean with a WZ Neo-male decapod crustacean. In one embodiment, thepresent invention provides a method for obtaining an all-WW femaleprogeny, comprising the step of mating a WW Neo-male decapod crustaceanwith a WW female decapod crustacean. In one embodiment, the presentinvention provides a method for obtaining an all-female progeny,comprising the step of mating a WW Neo-male decapod crustacean with a WZfemale decapod crustacean. In one embodiment, the present inventionprovides a method for obtaining a WW Neo-male decapod crustacean,comprising the step of injecting to a decapod crustacean comprising theWW sex chromosomes and younger than 180 days post-larva, a compositioncomprising at least 1×10² solitary cells derived from an androgenicgland (AG) of a decapod crustacean, thereby obtaining a WW Neo-maledecapod crustacean. In another embodiment, injecting is injecting intothe muscular tissue of an abdominal segment. In another embodiment,androgenic gland of a decapod crustacean is a hypertrophied androgenicgland of a decapod crustacean.

In another embodiment, the invention provides the progeny of a crossingbetween a WW decapod crustacean and another decapod crustacean. Inanother embodiment, the invention provides that another decapodcrustacean is a decapod crustacean having natural sex chromosomes(naturally occurring in nature). In another embodiment, the inventionprovides that another decapod crustacean is a wild-type decapodcrustacean. In another embodiment, the invention provides that anotherdecapod crustacean is a decapod crustacean manipulated according to themethods described herein such as a Neo-male. In another embodiment, theinvention provides that another decapod crustacean is a progeny of adecapod crustacean manipulated according to the methods describedherein. In another embodiment, the WW decapod crustacean is a male or aNeo-male. In another embodiment, the WW decapod crustacean is a female.In another embodiment, the invention provides a female decapodcrustacean obtained by the methods as described herein. In anotherembodiment, the invention provides a Neo-male decapod crustacean derivedfrom a WW female obtained by the methods as described herein.

In another embodiment, Neo-males are produced by manipulating theprogeny resulting from crossing sexually matured male (ZZ) and female(WZ) of the decapod crustacean (P crossing). In another embodiment, theprogeny of male (ZZ) and female (WZ) crossing is the post larvae (PL)comprised of about 50% males and about 50% females (progeny of Pcrossing).

In another embodiment, prior to PL₁₂₀, identified female PLs derivedfrom progeny of P crossing were manipulated by injecting a compositioncomprising cells as described herein for obtaining WZ Neo-males. Inanother embodiment, a sexually mature WZ Neo-male was crossed with awild-type female or a WZ female (F1 crossing). In another embodiment,the progeny of this F1 cross yielded about 3:1 female to male sex ratio.In another embodiment, ZZ male progeny of this F1 crossing were excludedand of the remaining females progeny of this F1 crossing, about 33% wereWW females. In another embodiment, using the W-specific DNA sex markerZZ males progeny of this F1 crossing were excluded and of the remainingfemales progeny of this F1 crossing, about 33% were identified as WW byusing a negative screen based on a Z-specific DNA sex marker.

In another embodiment, WW (genetic females) PLs of the F2 were furthermanipulated prior to PL₁₂₀ by injecting a composition comprising cellsas described herein for obtaining WW Neo-males. In another embodiment,WW Neo-males were grown until male-specific secondary sexualcharacteristics such as AM and gonopores at the base of the fifthwalking legs were verified.

In another embodiment, the present invention further provides crossingWW females and WW Neo-males. In another embodiment, the presentinvention further provides a method for obtaining multiple generationsof an all-female progeny by crossing WW females and WW Neo-males. Inanother embodiment, an all-female progeny is a progeny consisting 100%females. In another embodiment, the present invention further provides amethod for obtaining an all-female progeny by crossing WW decapodcrustacean with another decapod crustacean or a WZ Neo-male. In anotherembodiment, the present invention further provides a method forobtaining an all-female progeny by crossing WW female decapod crustaceanwith another decapod crustacean. In another embodiment, the presentinvention further provides a method for obtaining an all-female progenyby crossing WW Neo-male decapod crustacean with a natural female or a WZfemale decapod crustacean. In another embodiment, natural is wild-type.

In another embodiment, the present invention further provides a methodfor obtaining an all-female progeny by crossing WW female decapodcrustacean with a ZZ male decapod.

In another embodiment, the present invention provides that cells of theinvention are derived from a primary cell culture. In anotherembodiment, the present invention provides a primary cell culturecomprising: cell culture medium and cells derived from an androgenicgland (AG) of a decapod crustacean. In another embodiment, the presentinvention provides a primary cell culture comprising: cell culturemedium and cells derived from a hypertrophied androgenic gland (AG) of adecapod crustacean. In another embodiment, “a primary cell culture” is acell culture derived from an androgenic gland. In another embodiment, “aprimary cell culture” according to the invention does not includeimmortalized cells. In another embodiment, “a primary cell culture”according to the invention does not include transformed cells. Inanother embodiment, “a primary cell culture” according to the inventiondoes not include neoplastic cells. In another embodiment, a compositionof the invention comprises “a primary cell culture”. According toexemplary embodiment, the present invention provides that a decapodcrustacean is Macrobrachium rosenbergii.

In another embodiment, at least 50% of the cells within a primary cellculture are solitary cells. In another embodiment, at least 60% of thecells within a primary cell culture are solitary cells. In anotherembodiment, at least 70% of the cells within a primary cell culture aresolitary cells. In another embodiment, at least 80% of the cells withina primary cell culture are solitary cells. In another embodiment, atleast 90% of the cells within a primary cell culture are solitary cells.In another embodiment, at least 95% of the cells within a primary cellculture are solitary cells. In another embodiment, cells within “aprimary cell culture” comprise or consist enzymatically-dissociatedandrogenic gland cell (or a solitary cell). In another embodiment, aprimary cell culture of the invention is utilized for an unexpectedlyrobust and successful method for producing a Neo-male decapod crustacean(a male producing the W and Z gametes or a male producing only Wgametes). In another embodiment, injecting a composition of theinvention into post-larvae female decapod crustacean results insex-reversal. In another embodiment, injecting a composition of theinvention into post-larvae WW or WZ female decapod crustacean results insex-reversal. In another embodiment, injecting a composition of theinvention comprising cells is transplanting cells as described herein.In another embodiment, injecting a composition of the inventioncomprising cells is transplanting solitary cells. In another embodiment,the source of the cells described herein and the decapod crustacean tobe treated with the cells described herein are of the same species. Inanother embodiment, a post-larvae female decapod crustacean (havingfemale chromosomes composition) which underwent sex reversal to asexually functional male—termed “Neo-male” is crucial for the laterproduction of mono-sex all-female progeny or culture.

In another embodiment, a Neo-male is an organism bearing the sexchromosome combination of a female (WZ or WW) and the sexual ability toreproduce as a male. In another embodiment, a Neo-male is an organismbearing the sex chromosome combination of a female (WZ or WW) andmale-functioning reproductive organs. In another embodiment, a Neo-maleis an organism bearing the sex chromosome combination of a female (WZ orWW) and male-functioning sex organs. In another embodiment, a Neo-maleproduces W-only gametes. In another embodiment, a Neo-male produces Wand Z gametes. In another embodiment, a Neo-male is an organism bearingthe sex chromosome combination of a female (WZ or WW) that: (a) wasmanipulated with the cells of the invention; and (b) upon sexualmaturity has/had the sexual ability to reproduce as a male.

In another embodiment, at least 40% of the genetic females undergoneinjection/transplantation of cells as described herein survived thisprocedure. In another embodiment, at least 50% of the genetic femalesundergone injection/transplantation of cells as described hereinsurvived this procedure. In another embodiment, at least 60% of thegenetic females undergone injection/transplantation of cells asdescribed herein survived this procedure. In another embodiment, atleast 70% of the genetic females undergone injection/transplantation ofcells as described herein survived this procedure. In anotherembodiment, at least 75% of the genetic females undergoneinjection/transplantation of cells as described herein survived thisprocedure. In another embodiment, at least 80% of the genetic femalesundergone injection/transplantation of cells as described hereinsurvived this procedure. In another embodiment, according to the presentexperiment 80% of the genetic females undergoneinjection/transplantation of cells as described herein survived thisprocedure (data not shown). In another embodiment, according to thepresent experiment 90% of the genetic females undergoneinjection/transplantation of cells as described herein survived thisprocedure (data not shown). In another embodiment, transplantationaccording to the invention is allogeneic transplantation.

In another embodiment, at least 40% to 90% of the genetic femalesundergone injection/transplantation of cells as described hereinsurvived this procedure. In another embodiment, at least 50% to 80% ofthe genetic females undergone injection/transplantation of cells asdescribed herein survived this procedure. In another embodiment, atleast 60% to 80% of the genetic females undergoneinjection/transplantation of cells as described herein survived thisprocedure. In another embodiment, at least 80% of the genetic femalesundergone injection/transplantation of cells as described hereinsurvived this procedure. In another embodiment, at least 85% of thegenetic females undergone injection/transplantation of cells asdescribed herein survived this procedure. In another embodiment, atleast 90% of the genetic females undergone injection/transplantation ofcells as described herein survived this procedure. In anotherembodiment, 80% to 95% of the genetic females undergoneinjection/transplantation of cells as described herein survived thisprocedure. In another embodiment, at least 20% of the surviving geneticfemales undergone injection/transplantation of cells as described hereindeveloped into Neo-males. In another embodiment, at least 30% of thesurviving genetic females undergone injection/transplantation of cellsas described herein developed into Neo-males. In another embodiment, atleast 45% of the surviving genetic females undergoneinjection/transplantation of cells as described herein developed intoNeo-males. In another embodiment, at least 50% of the surviving geneticfemales undergone injection/transplantation of cells as described hereindeveloped into Neo-males. In another embodiment, at least 60% of thesurviving genetic females undergone injection/transplantation of cellsas described herein developed into Neo-males. In another embodiment, atleast 70% of the surviving genetic females undergoneinjection/transplantation of cells as described herein developed intoNeo-males. In another embodiment, at least 80% of the surviving geneticfemales undergone injection/transplantation of cells as described hereindeveloped into Neo-males.

In another embodiment, at least 25% of the genetic females undergoneinjection/transplantation of cells as described herein developed intoNeo-males. In another embodiment, a “Neo-male” is a genetic femalehaving the capability of fertilizing eggs In another embodiment, a“Neo-male” is a genetic female having and/or producing sperm cells. Inanother embodiment, “male” comprises “Neo-male”. In another embodiment,a “Neo-male” is a genetic female having/producing spermatogonium. Inanother embodiment, a “Neo-male” is a genetic female having/producingspermatozoa. In another embodiment, a “Neo-male” is a genetic femalehaving sperm cells, wherein the sperm cells are capable of fertilizingeggs. In another embodiment, having the capability of fertilizing eggsis having the capability to mate with a female decapod crustacean asdescribed herein. In another embodiment, a “Neo-male” is a geneticfemale having the capability of fertilizing eggs upon sexual maturityinto a male. In another embodiment, a “Neo-male” is a genetic femalecomprising Appendix masculina (AM). In another embodiment, a “Neo-male”is a genetic female comprising at least one male gonopore. In anotherembodiment, a “Neo-male” is a genetic female comprising functionallyactive masculine gonad (i.e., testis). In another embodiment, a“Neo-male” is a genetic female comprising Appendix masculina (AM), atleast one male gonopore, a masculine gonad and having the capability offertilizing eggs upon sexual maturity. In another embodiment, at least30% of the genetic females undergone injection/transplantation of cellsas described herein developed into Neo-males. In another embodiment, atleast 35% of the genetic females undergone injection/transplantation ofcells as described herein developed into Neo-males. In anotherembodiment, at least 40% of the genetic females undergoneinjection/transplantation of cells as described herein developed intoNeo-males. In another embodiment, at least 45% of the genetic femalesundergone injection/transplantation of cells as described hereindeveloped into Neo-males. In another embodiment, at least 50% of thegenetic females undergone injection/transplantation of cells asdescribed herein developed into Neo-males. In another embodiment, atleast 60% of the genetic females undergone injection/transplantation ofcells as described herein developed into Neo-males. In anotherembodiment, at least 70% of the genetic females undergoneinjection/transplantation of cells as described herein developed intoNeo-males. In another embodiment, at least 80% of the genetic femalesundergone injection/transplantation of cells as described hereindeveloped into Neo-males. In another embodiment, at least 90% of thegenetic females undergone injection/transplantation of cells asdescribed herein developed into Neo-males.

In another embodiment, 25% to 90% of the genetic females undergoneinjection/transplantation of cells as described herein developed intoNeo-males. In another embodiment, 25% to 60% of the genetic femalesundergone injection/transplantation of cells as described hereindeveloped into Neo-males. In another embodiment, 25% to 50% of thegenetic females undergone injection/transplantation of cells asdescribed herein developed into Neo-males.

In another embodiment, mating a Neo-male with a natural (such as awild-type) female resulted in a progeny comprising at least 500individual WW females. In another embodiment, mating a Neo-male with anatural female resulted in a progeny comprising at least 600 individualWW females. In another embodiment, mating a Neo-male with a naturalfemale resulted in a progeny comprising at least 700 individual WWfemales. In another embodiment, mating a Neo-male with a natural femaleresulted in a progeny comprising at least 800 individual WW females. Inanother embodiment, mating a Neo-male with a natural female resulted ina progeny comprising at least 900 individual WW females. In anotherembodiment, mating a Neo-male with a natural female resulted in aprogeny comprising at least 1,000 individual WW females. In anotherembodiment, mating a Neo-male with a natural female resulted in aprogeny comprising 150 to 1,500 individual WW females. In anotherembodiment, mating a Neo-male with a natural female resulted in aprogeny comprising 200 to 2,000 individual WW females. In anotherembodiment, mating a Neo-male with a natural female resulted in aprogeny comprising 250 to 2,500 individual WW females. In anotherembodiment, the number of WW offspring was determined based on multiplesampling of multiple crossings (data not shown).

In another embodiment, mating a WW Neo-male with a WW female resulted ina progeny comprising 500 to 5,000 individual WW females. In anotherembodiment, mating a WW Neo-male with a WW female resulted in a progenycomprising 1000 to 10,000 individual WW females. In another embodiment,mating a WW Neo-male with a WW female resulted in a progeny comprising2,000 to 50,000 individual WW females. In another embodiment, mating aWW Neo-male with a WW female resulted in a progeny consisting 100%females. In another embodiment, mating a WW Neo-male with a WW femaleresulted in a progeny consisting 100% WW females.

In another embodiment, crossing a WW Neo-male with a natural femaleresulted in a progeny consisting 100% females. In another embodiment,crossing a WZ Neo-male with a WW female resulted in a progeny consisting100% females. In another embodiment, crossing a ZZ male with a WW femaleresulted in a progeny consisting 100% females.

In another embodiment, a “Neo-male” is a decapod crustacean Neo-male. Inanother embodiment, a “male” is a decapod crustacean male. In anotherembodiment, a “female” is a decapod crustacean female.

In another embodiment, the present invention provides a method forgenerating a WW female. In another embodiment, the present inventionprovides a method for generating a WW Neo-male. In another embodiment, aWW female cannot be a product of nature in species where intersexualityand/or hermaphroditism occurs unnaturally. In another embodiment, thepresent invention provides a WW female. In another embodiment, thepresent invention provides a method for generating a WW femalecomprising crossing a WZ Neo-male (a manipulated sexually reversedfemale) with a natural female or a WZ female. In another embodiment, thepresent invention provides that WZ Neo-male are brothers (offspring of asingle crossing between natural ZZ male and WZ female-P generation). Inanother embodiment, W or Z refers to the sex chromosome or a gamete of adecapod crustacean as described herein. In another embodiment, WW, WZ orZZ refers to the genotypic sex of a decapod crustacean as describedherein.

In another embodiment, Neo-males are produced by manipulating theprogeny resulting from crossing sexually matured male (ZZ) and female(WZ) of the decapod crustacean (P crossing). In another embodiment,female PLs derived from progeny of P crossing were segregated frommales. In another embodiment, female PLs derived from progeny of Pcrossing were segregated from males by means of molecular genetic sexdetermination using a W-specific DNA sex marker.

In another embodiment, prior to PL₁₂₀, identified female PLs derivedfrom progeny of P crossing were manipulated by injecting a compositioncomprising cells as described herein for obtaining WZ Neo-males. Inanother embodiment, WZ Neo-males were grown until male-specificsecondary sexual characteristics such as development of Appendixmasculina (AM) and gonopores at the base of the fifth walking legs wereverified.

In another embodiment, 2 to 250 siblings having 2 types of gametes, ˜50%bearing the W sex chromosome and ˜50% bearing the Z sex chromosome ofthe F1 generation are randomly chosen for sex-reversal manipulation. Inanother embodiment, at least about 50% of the siblings having 2 types ofgametes, ˜50% bearing the W sex chromosome and ˜50% bearing the Z sexchromosome of the F1 generation that were randomly chosen forsex-reversal manipulation actually mature into Neo-males. In anotherembodiment, Neo-males (sibling) that were grown to sexual maturity werefurther crossed with natural WZ females. In another embodiment, providedherein a method for obtaining a WW female comprising crossing Neo-maleswith natural WZ females (F2 progeny).

In another embodiment, 2 to 150 female WW siblings as described hereinare randomly chosen for sex-reversal manipulation and for generating aWW Neo-male. In another embodiment, at least about 50% of the female WWsiblings which were randomly chosen for sex-reversal manipulationactually mature into WW Neo-males. In another embodiment, WW Neo-malesthat were grown to sexual maturity were further crossed with natural WZfemales or WW females.

In another embodiment, provided herein a method for obtaining a WWfemale comprising crossing a Neo-male with a natural WZ female (F2progeny) and obtaining a population with mixed sex genotypes whichincludes a WW female population. In another embodiment, provided hereina method for obtaining a WW Neo-male comprising: (1) crossing a Neo-malewith a natural WZ female (F2 progeny) and obtaining a population withmixed sex genotypes which includes a WW female; and (2) administrating acomposition comprising cells of the invention according to the methodsof generating a Neo-male as disclosed herein, thereby generating and/orobtaining a WW Neo-male.

In another embodiment, F2 progeny (the progeny of Neo-males and WZfemales) gave rise to at least 500 WW females. In another embodiment, F2progeny (the progeny of Neo-males and WZ females) gave rise to at least700 WW females. In another embodiment, F2 progeny (the progeny ofNeo-males and WZ females) gave rise to at least 900 WW females. Inanother embodiment, F2 progeny (the progeny of Neo-males and WZ females)gave rise to at least 1,000 WW females. In another embodiment, F2progeny (the progeny of Neo-males and WZ females) gave rise to 200 to2,000 WW females. In another embodiment, F2 progeny (the progeny ofNeo-males and WZ females) gave rise to 250 to 2,500 WW females. Inanother embodiment, F2 progeny (the progeny of Neo-males and WZ females)gave rise to at least 1,200 WW females. In another embodiment, at leastfive WW females are cousins. In another embodiment, the cousins WWfemales are produced from crossings involving five different Neo-males,wherein the Neo-males are brothers originating from the same parents.

In another embodiment, F2 progeny is further manipulated by the methodsdescribed herein for the generation of a WW Neo-male. In anotherembodiment, about 40% to 80% of the WW females undergone sex reversalmanipulation actually mature to sexually reproductive WW Neo-males. Inanother embodiment, about 50% to 60% of the WW females undergone sexreversal manipulation actually mature to sexually reproductive WWNeo-males.

In another embodiment, all cells within the primary cell culture arecells derived from an androgenic gland (AG) of a decapod crustacean. Inanother embodiment, the primary cell culture is an “androgenic glandprimary cell culture or a “hypertrophied androgenic gland primary cellculture”. In another embodiment, cell injection and/or transplantationis allogeneic transplantation of cells.

In another embodiment, at least 50% of the cells within the primary cellculture and/or the composition as described herein are endocrine AGcells. In another embodiment, at least 60% of the cells within theprimary cell culture and/or the composition as described herein areendocrine AG cells. In another embodiment, at least 65% of the cellswithin the primary cell culture and/or the composition as describedherein are endocrine AG cells. In another embodiment, at least 70% ofthe cells within the primary cell culture and/or the composition asdescribed herein are endocrine AG cells. In another embodiment, at least75% of the cells within the primary cell culture and/or the compositionas described herein are endocrine AG cells. In another embodiment, atleast 80% of the cells within the primary cell culture and/or thecomposition as described herein are endocrine AG cells. In anotherembodiment, at least 85% of the cells within the primary cell cultureand/or the composition as described herein are endocrine AG cells. Inanother embodiment, at least 90% of the cells within the primary cellculture and/or the composition as described herein are endocrine AGcells. In another embodiment, at least 95% of the cells within theprimary cell culture and/or the composition as described herein areendocrine AG cells. In another embodiment, at least 98% of the cellswithin the primary cell culture and/or the composition as describedherein are endocrine AG cells. In another embodiment, at least 99% ofthe cells within the primary cell culture and/or the composition asdescribed herein are endocrine AG cells. In another embodiment,endocrine AG cells are endocrine solitary AG cells as described herein.

In another embodiment, at least 8% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 10% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 20% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 25% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 30% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 35% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 40% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 45% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 50% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 60% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 70% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 80% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 90% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.In another embodiment, at least 95% of the cells within the primary cellculture and/or the composition as described herein are solitary cells.

In another embodiment, at least 8% of the cells within the primary cellculture and/or the composition as described herein are aggregated. Inanother embodiment, at least 10% of the cells within the primary cellculture and/or the composition as described herein are agglomerate. Inanother embodiment, at least 20% of the cells within the primary cellculture and/or the composition as described herein are agglomerate. Inanother embodiment, at least 25% of the cells within the primary cellculture and/or the composition as described herein are agglomerate. Inanother embodiment, at least 30% of the cells within the primary cellculture and/or the composition as described herein are aggregate. Inanother embodiment, at least 35% of the cells within the primary cellculture and/or the composition as described herein are aggregate. Inanother embodiment, at least 40% of the cells within the primary cellculture and/or the composition as described herein are aggregate. Inanother embodiment, at least 45% of the cells within the primary cellculture and/or the composition as described herein are aggregate. Inanother embodiment, at least 50% of the cells within the primary cellculture and/or the composition as described herein are aggregate. Inanother embodiment, at least 60% of the cells within the primary cellculture and/or the composition as described herein are aggregate. Inanother embodiment, at least 70% of the cells within the primary cellculture and/or the composition as described herein are aggregate.

In another embodiment, an aggregate of cells of the invention comprises2 to 70 cells. In another embodiment, an aggregate of cells of theinvention comprises 2 to 50 cells. In another embodiment, an aggregateof cells of the invention comprises 2 to 40 cells. In anotherembodiment, an aggregate of cells of the invention comprises 2 to 30cells. In another embodiment, an aggregate of cells of the inventioncomprises 2 to 25 cells. In another embodiment, an aggregate of cells ofthe invention comprises 2 to 20 cells.

In another embodiment, the primary cell culture and/or the compositionas described herein is devoid or substantially devoid of an intacttissue. In another embodiment the primary cell culture and/or thecomposition as described herein is devoid or substantially devoid oftissue fragments.

In another embodiment, an hypertrophied AG gland is also ahyperplastic/hyperplasic AG gland. In another embodiment, ahypertrophied AG gland is a gland which its volume was increased. Inanother embodiment, a hypertrophied AG gland is a gland which its volumewas increased by artificial means. In another embodiment, ahypertrophied AG gland is a gland with enlarged (compared to an intactgland) component cells. In another embodiment, a hyperplasic AG ischaracterized by an increased number of cells (compared to an intactgland).

In another embodiment, a primary cell culture and/or a composition asdescribed herein comprises at least 1×10² cells or solitary cellsderived from an AG of a decapod crustacean. In another embodiment, aprimary cell culture and/or a composition as described herein comprisesat least 1×10³ cells or solitary cells derived from an AG of a decapodcrustacean. In another embodiment, a primary cell culture and/or acomposition as described herein comprises at least 1×10⁴ cells orsolitary cells derived from an AG of a decapod crustacean. In anotherembodiment, a primary cell culture and/or a composition as describedherein comprises at least 1×10⁵ cells or solitary cells derived from anAG of a decapod crustacean. In another embodiment, a primary cellculture and/or a composition as described herein comprises at least1×10⁶ cells or solitary cells derived from an AG of a decapodcrustacean. In another embodiment, a primary cell culture and/or acomposition as described herein comprises at least 1×10⁷ cells orsolitary cells derived from an AG of a decapod crustacean. In anotherembodiment, a primary cell culture and/or a composition as describedherein comprises at least 1×10⁸ cells or solitary cells derived from anAG of a decapod crustacean.

In another embodiment, a composition as describe herein comprises cellculture medium. In another embodiment, cell culture medium supports thegrowth of the cells described herein. In another embodiment, cellculture medium supports the proliferation of the cells described herein.In another embodiment, cell culture medium induces or maintainsandrogenic hormone secretion by the cells described herein. In anotherembodiment, suitable cell culture mediums are known to one of skill inthe art.

In another embodiment, a composition as describe herein comprises:Arginine, biotin, NaCl, glucose, insulin, Cystine, choline, KCl,penicillin, transferrin, Glutamine, folate, NaH₂PO₄, streptomycin,specific growth factors, Histidine nicotinamide, NaHCO₃, phenol red,Isoleucine, pantothenate, CaCl₂, whole serum, Leucine, pyridoxal, MgCl₂,Lysine, thiamine, Methionine, riboflavin, Phenylalanine, Threonine,Trytophan, Tyrosine, Valine or any combination thereof.

In another embodiment, suitable cell culture medium comprises L-15. Inanother embodiment, suitable cell culture medium comprises bufferedand/or osmolality-adjusted L-15. In another embodiment, suitable cellculture medium comprises one or more free base amino acid. In anotherembodiment, suitable cell culture medium comprises L-Glutamine. Inanother embodiment, suitable cell culture medium comprises serum. Inanother embodiment, suitable cell culture medium comprises fetal bovineserum. In another embodiment, suitable cell culture medium comprises anantibiotic or a combination of antibiotic such as but not limited toPenicillin-Streptomycin.

Process

In another embodiment, provided herein a method for obtainingenzymatically-dissociated AG primary cell culture in suspension fromdecapod crustaceans. In another embodiment, the invention provides aprocess for obtaining a primary cell culture, wherein the primary cellculture comprises/consists cells derived from an AG of a decapodcrustacean and medium, comprising the step of: Obtaining solitary cellsby enzymatically-dissociating AG cells from a AG of a decapodcrustacean; thereby obtaining a primary cell culture comprising cellculture medium and cells derived from an AG of a decapod crustacean. Inone embodiment, androgenic gland is a hypertrophied androgenic gland.

In another embodiment, the invention provides a process for obtaining aprimary cell culture, wherein the primary cell culturecomprises/consists cells derived from an AG of a decapod crustacean andmedium, comprising the steps: (a) Obtaining solitary cells byenzymatically-dissociating AG cells from an AG of a decapod crustacean;optionally (b) Seeding the disassociated AG cells in a cell culturemedium; and optionally (c) Incubating the cells from step (b) in anincubator at 25° C. to 27° C.; thereby obtaining a primary cell culturecomprising cell culture medium and cells derived from an AG of a decapodcrustacean. In another embodiment, an incubator is a CO₂-free incubator.In another embodiment, the incubator's temperature is about 26° C. Inanother embodiment, the incubator's temperature is 25.5-26.5° C. Inanother embodiment, the incubator's temperature is about 25-27° C.

Sexually Reversed Decapods

In another embodiment, the present invention provides a decapodcrustacean, comprising the primary cell culture and/or the compositionas described herein. In another embodiment, the present inventionprovides a female decapod crustacean, comprising the primary cellculture and/or the composition as described herein. In anotherembodiment, a decapod crustacean or a female decapod crustacean is up to180 days post-larvae old decapod crustacean. In another embodiment, thepresent invention provides a female decapod crustacean undergoing sexreversal, comprising the primary cell culture and/or the composition asdescribed herein. In another embodiment, the present invention providesan offspring of the female decapod crustacean that underwent sexreversal according to the present invention. In another embodiment, thepresent invention provides a homogametic WW decapod crustacean offspringof the female decapod crustacean that underwent sex reversal accordingto the present invention. In another embodiment, the present inventionfurther provides a homogametic WW Neo-male decapod crustacean. Inanother embodiment, the present invention further provides a homogameticWW Neo-male decapod crustacean derived from a WW female as describedherein.

In one embodiment, WW homogametic male is a male having only the W sexchromosome in its sex gametes. In one embodiment, WW homogametic male isa male devoid of Z sex chromosome in its gametes. In one embodiment, WWhomogametic male is a male consisting W sex chromosome in its sexgametes.

In another embodiment, the present invention provides a method forobtaining an all-female progeny, comprising the step of mating ahomogametic WW female with a male decapod crustacean, thereby obtainingan all-female progeny. In another embodiment, the present inventionprovides a method for obtaining an all-female progeny, comprising thestep of mating a homogametic WW female with a homogametic WW Neo-maledecapod crustacean, thereby obtaining an all-female progeny. In anotherembodiment, the present invention provides a method for obtaining anall-female progeny, comprising the step of mating a homogametic WWfemale with a heterogametic male decapod crustacean, thereby obtainingan all-female progeny. In another embodiment, the present inventionprovides a method for obtaining an all-female progeny, comprising thestep of mating a homogametic WW Neo-male with a heterogametic femaledecapod crustacean, thereby obtaining an all-female progeny. In anotherembodiment, the present invention provides a method for obtaining anall-female progeny, comprising the step of mating a homogametic WWfemale with a homogametic male decapod crustacean, thereby obtaining anall-female progeny. In another embodiment, the present inventionprovides a method for obtaining an all-female progeny, comprising thestep of mating a WW Neo-male and WW female thus obtaining 100% all WWfemale progeny. In another embodiment, the present invention provides amethod for obtaining an all-female progeny, comprising the step ofmating a ZZ male and WW female thus obtaining 100% all WZ femaleprogeny. In another embodiment, the present invention provides a methodfor obtaining an all-female progeny, comprising the step of mating a WZNeo male and WW female thus obtaining 50% WZ female and 50% WW femaleprogeny.

In another embodiment, a homogametic WW female is an offspring of thefemale decapod crustacean that underwent sex reversal according to thepresent invention and developed into a Neo-male (sexually functionalmale). In another embodiment, a homogametic WW Neo-male is a previous WWfemale decapod crustacean that underwent sex reversal according to thepresent invention.

In another embodiment, the present invention provides a method forobtaining a male having the W sex chromosome (WW or WZ), comprising thestep of injecting to a decapod crustacean comprising the W sexchromosome and younger than 180 days post-larva, a compositioncomprising at least 1×10² solitary cells derived from an AG of a decapodcrustacean, thereby obtaining a male having the W sex chromosome. Inanother embodiment, the present invention provides a method forobtaining a male having the W sex chromosome (genetically a female),comprising the step of injecting to a genetically female decapodcrustacean (comprising the W sex chromosome) younger than 180 dayspost-larva, a composition comprising at least 1×10² solitary cellsderived from an AG of a decapod crustacean.

In another embodiment, younger than 180 days post-larva is younger than160 days post-larva. In another embodiment, younger than 180 dayspost-larva is younger than 150 days post-larva. In another embodiment,younger than 180 days post-larva is younger than 130 days post-larva. Inanother embodiment, younger than 180 days post-larva is younger than 120days post-larva. In another embodiment, younger than 180 days post-larvais younger than 100 days post-larva. In another embodiment, younger than180 days post-larva is younger than 80 days post-larva. In anotherembodiment, younger than 180 days post-larva is younger than 60 dayspost-larva. In another embodiment, younger than 180 days post-larva is1-60 days post-larva. In another embodiment, younger than 180 dayspost-larva is younger than 30 days post-larva.

In another embodiment, injecting is injecting into the muscular tissueof an abdominal segment. In another embodiment, injecting is injectinginto the muscular tissue of the first abdominal segment. In anotherembodiment, injecting is injecting into the muscular tissue of thesecond abdominal segment. In another embodiment, injecting is allogeneiccell transplantation into the muscular tissue of an abdominal segment.

In another embodiment, provided herein a method for injection ofenzymatically-dissociated AG primary cell culture in suspension indecapod crustaceans. In another embodiment, provided herein a method forproduction of Neo-male decapod crustaceans by injection of dissociatedAG cells obtained from primary culture in suspension—the composition. Inanother embodiment, provided herein a method for production of Neo-maledecapod crustaceans by a single injection of dissociated AG cellsobtained from primary culture in suspension.

In another embodiment, the cells are obtained from a hypertrophied AG ofa male decapod crustacean following at least 5 days from bi-lateralsurgical removal of the X-organ Sinus-gland complex. In anotherembodiment, the cells are obtained from a hypertrophied AG of a maledecapod crustacean following at least 8 days from bi-lateral surgicalremoval of the X-organ Sinus-gland complex. In another embodiment, thecells are obtained from a hypertrophied AG of a male decapod crustaceanfollowing any known treatment that can induce hypertrophy in an AG. Inanother embodiment, the cells are obtained from a hypertrophied AG of amale decapod crustacean following 4-15 days from bi-lateral surgicalremoval of the X-organ Sinus-gland complex.

In another embodiment, provided herein the composition described hereinis injected once or more into a female decapod crustacean at the agerange from 1 to 100 days post-larvae. In another embodiment, providedherein the composition described herein is injected once or more into afemale decapod crustacean at the age range from 1 to 50 dayspost-larvae. In another embodiment, provided herein the compositiondescribed herein is injected once or more into a female decapodcrustacean at the age range from 7 to 30 days post-larvae. In anotherembodiment, injection is by means of a micro-injector apparatus. Inanother embodiment, injection to a sexually immature female inducesfunctional sex reversal to male and/or Neo-male. The latter male and/orNeo-male is used, in some embodiments, to obtain all-female mono-sexpopulation by producing WW females.

As used herein, the singular forms “a”, “an”, and “the” include pluralforms unless the context clearly dictates otherwise. Thus, for example,reference to “a therapeutic agent” includes reference to more than onetherapeutic agent.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

As used herein, the terms “comprises,” “comprising,” “containing,”“having” and the like can have the meaning ascribed to them in U.S.patent law and can mean “includes,” “including,” and the like;“consisting essentially of or “consists essentially” likewise has themeaning ascribed in U.S. patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments. In one embodiment, the terms “comprises,” “comprising,“having” are/is interchangeable with “consisting”.

In some embodiments, a composition of the invention comprisespharmaceutically active agents. In some embodiments, pharmaceuticallyactive agents are added prior to transplantation. Pharmaceuticallyactive agents include but are not limited to any of the specificexamples disclosed herein. Those of ordinary skill in the art willrecognize also numerous other compounds that fall within this categoryand are useful according to the invention.

As used herein, “an effective amount” refers to that amount of cellsthat produces the desired effect (such as treatment or sex-reversal).

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference. Other general references are provided throughout thisdocument.

Material and Methods Animals

Macrobrachium rosenbergii blue claw males (40±5 g) were reared inseparate 600 L tanks at 28±2° C. with constant aeration, light regime of14:10 (L:D) and fed ad libitum (shrimp pellets comprising 30% protein)in the facilities of Enzootic Holdings Ltd. M. rosenbergii youngindividuals post metamorphosis (i.e., post larvae (PL)) were reared in a3.5 cubic meter U-shaped tank, and maintained as above.

Androgenic Gland Hypertrophy and Enzymatic Cell Dissociation

The neuroendocrine X-organ Sinus-gland complex, located in the eyestalk,is known to secrete neuropeptides which negatively regulate theandrogenic gland (AG) and its activities. Thus, an endocrinemanipulation which involves its elimination via surgical removal andinduces both hypertrophy and hyperplasia of the AG was applied to the M.rosenbergii blue claw males. Eight days post the endocrine manipulation;the induced males were anesthetized for 15 minutes in ice cold watersupplemented with 0.2% hypochlorite for disinfection purposes.Thereafter, animals were dissected and their hypertrophied AGs wereisolated under dissecting light microscope. Subsequently, cells wereseparated by means of enzymatic dissociation. Briefly, 4-5 hypertrophiedAGs were pooled in a single tube and were placed on ice. Thereafter, 1ml of a specific enzyme mix and antibiotics [Leibovitz L-15 Medium withL-Glutamine, 0.1% weight/volume (w/v) Collagenase type I, 0.1% (w/v)Collagenase type IV and Penicillin-Streptomycin Solution] were added.The dissociation reaction tube was then placed in a rotator at a speedof 25 RPM for 40 minutes at room temperature (RT) and followed by acentrifugation at 500×g for 5 minutes, also at RT. Upon centrifugation,the tube was placed in a sterilized biological laminar flow hood. Theupper phase (does not include living cells) was removed and the cellpellet was washed by re-suspending it in 1 ml of feeding media[Leibovitz L-15 Medium with L-Glutamine, 10% volume/volume (v/v) fetalbovine serum and Penicillin-Streptomycin Solution] and subsequentcentrifugation at 500×g for 5 minutes at RT. Washing procedure wasrepeated three times. Finally, cells were re-suspended in 500 μl offeeding media.

Cell Counting

Cell concentration and viability were measured using standard Trypanblue stain. Briefly, cells were stained with Trypan blue solution at afinal concentration of 0.08% and then loaded to a hemocytometer forobservation under a light microscope at a magnification of ×100.

AG Primary Cell Culture

Fractions of 10 μl of the suspended cells in the media were eitherimmediately seeded in a 24-well plate coated with 20 μg/ml poly D-lysine(PDL) at a density of ˜1×10⁴ cells per well or first loaded into amicro-injector apparatus, passed through the micro-injector glasscapillary into a 1.5 ml tube and then seeded in a 24-well plate at adensity of ˜1×10⁴ cells per well. Of the collected cells, a fraction wasallocated for viability assessment and stained as described above. Thecells were grown in a CO₂-free incubator at 27° C. Twenty four hoursafter seeding and onwards, the growing media was partially replacedevery other day. Overall, cells were maintained for 21 days during whichthey were monitored under an inverted light microscope and theirmorphology, density and interactions were documented.

Injection of an AG Cell Suspension into PLs

The ability of purified and grown AG cells to induce sex reversal wasexamined. Mixed population (males and females) of PL 60 or 30 days orless (PL_(<60), PL_(<30); n=913) were injected with ˜2×10³ AG cells. Ingeneral, each PL was restrained on a plasticine surface and using themicro-injector apparatus, while observing through a laboratorybinocular, the AG cells from the primary culture were suspended andadministrated via a single injection into the muscular tissue of thefirst abdominal segment. Thereafter, the injected PLs were divided intotwo groups: (1) Comprising the majority of PLs (n=883) which were keptin either a U-shaped tank of 3.5 cubic meters or a circular tank of ˜10cubic meters or a rectangular (˜250 m²; ˜8 m×30 m) earthen pond with awater depth of 1 m for grow-out. (2) Comprising representative PLs(n=30) which were kept in a 3.5 cubic meter U-shaped tank. The latterPLs were routinely examined and followed-up for sex reversal intoNeo-males.

Appendix Masculina (AM) and Male Gonopores Examination

To evaluate masculine features, the 2^(nd) pleopod (swimming leg) wasremoved using fine tweezers and the presence/or lack of the AM wasconfirmed under a light microscope. In mature Neo-males (1 year of age)a regeneration assay was performed. Briefly, Neo-males with confirmedAM, as described above, were allowed to molt and the regeneration of AMwas examined.

The appearance of male gonopores at the base of the 5^(th) pereiopods(walking leg) were examined by placing the PL on its dorsal side andviewing under a laboratory binocular.

Molecular Analysis

The 2^(nd) pleopods previously removed for AM observation had been usedfor genomic DNA (gDNA) extraction for the genetic determination of sex.Briefly, gDNA was extracted using the REDExtract-N-Amp Tissue PCR Kit(Sigma, Israel) and used as a template for PCR of either a W- orZ-specific DNA sex markers (Ventura et al., 2011). PCR products wereseparated on a 2% agarose gel, stained with Ethidium Bromide andvisualized on a UV table.

Histology

Testes were dissected out together with the proximal sperm duct (‘vasdeferens’) from Neo-males (1 year of age). Tissue samples were fixed inmodified Carnoy's II [Ethanol 60% (v/v; Bio-Lab Ltd, Israel), Chloroform30% (v/v; Frutarom, Israel), Glacial acetic acid 10% (v/v; Sigma,Israel) and Formaldehyde 2% (v/v; Sigma, Israel) for 24 h, dehydratedgradually through a series of increasing alcohol concentrations up toXylen (Sigma, Israel) and embedded in Paraplast (Kendall, Mansfield,Mass.) according to conventional procedures. Sections of 5 μm were cutonto silane-coated slides (Menzel-Glaser, Braunschweig, Germany).Consecutive sections were stained using standard Hematoxylin (Bar Naor,Israel) and Eosin (Bar Naor, Israel) for morphological observations.

Example 1 Production of WZ Neo-Males Using a Single Injection of theCell Suspension into Post Larvae Female Decapod Crustacean

The experimental data provided herein demonstrates the ability toproduce an enzymatically-dissociated decapod crustaceans androgenicgland primary cell culture in suspension and the utilization of thedecapod crustaceans androgenic gland primary cell culture for theproduction of Neo-males. These Neo-males are further utilized for matingand producing a WW females resulting in the generation of a mono-sexall-female culture-progeny.

W and Z Chromosomes—Sex Determination

The schematic flow diagram presented in FIG. 1 illustrates the W and Zchromosome-based sex determination model. According to this genetic modeof inheritance, females are heterogametic (WZ) and males are homogametic(ZZ). Phase 1 of the diagram highlights that even a single injection ofan AG primary cell culture in suspension to young decapod crustaceanfemale PLs can induce a full and functional sex reversal into a WZNeo-male. Unexpectedly, this manipulation did not cause any illnesses ordeath to the treated animals. It is important to emphasize the presentinvention overcomes the hurdles associated with AG tissue implantationwhich resulted in devastating death rate and low sex reversal rate.

The “produced” WZ Neo-males, which are fully sexually functional asmales, are genetic females bearing a W gamete. Upon a successful matingof a Neo-male (WZ) with a natural female (such as WZ), 25% of theprogeny were found to be males (ZZ) and the remaining 75% were females.Out of the latter, 67% were identified by means of molecular genotypingas natural heterogametic (WZ) females while the remaining 33% were foundto be unique WW homogametic females. Further mating these WW homogameticfemales with ZZ males gave rise to a 100% WZ heterogametic femaleprogeny.

Phase 2 of the diagram highlights that a single AG cell suspensioninjection to a PL WW female (of phase 1) results with a fully functionalsex reversed WW Neo-male. Incrossing of a WW homogametic male/Neo-malewith a homogametic female gave rise to a 100% WW homogametic progeny.

As a prerequisite to obtain a suspensible hypertrophied androgenic glandcell culture, male decapod crustaceans must beendocrinologically-induced so the levels of specific AG-inhibitingneuropeptides are decreased and the gland produces sufficient amounts ofandrogenic factors that can actually induce development of male in ananimal capable of developing to both male and female (regardless of itssex chromosomes composition). This intervention, in turn, leads to thehypertrophy of the AG and its comprising cells.

Histological comparisons between intact AG (FIG. 2A) and a hypertrophiedAG (FIG. 2B) clearly demonstrate the substantial differences in thetotal size of the AG, the number of cells comprising it and theircharacterization. Surgically removed hypertrophied AGs were dissociatedusing a specific enzymatic mixture to obtain a suspension of individualcells.

The passage through the micro-injector did not seem to have an apparenteffect on the viability of the AG cells. The seeded primary cell culturewas documented on days 2 and 6 (FIGS. 3A and 3B, respectively), and wasfound to be viable for as long as 21 days in culture.

Light microscope images of the culture highlighted the viability ofcells, demonstrating well-developed cell extensions (FIG. 3). The latterare hallmark characterizations of a healthy living primary culture, asoppose to dead cells which tend to shrink and subsequently detach fromthe well surface and float.

In order to inject the cell suspension to very small PLs, at early agesas 20 to 60 days or less post metamorphosis (termed PL_(<60)), aspecialized infrastructure combining a micro-injector apparatus and alaboratory binocular, was assembled (FIG. 4). The manipulation wasperformed on each restrained animal under a laboratory binocular using amicro-injector apparatus (FIG. 4).

A total of 913 PLs were injected once with an AG primary cell culturesuspension, with a PL survival rate of 81.

When reaching sexual maturity, M. rosenbergii demonstrates clear sexualdimorphism and males are highly distinct from females (FIG. 5A). Priorto full sexual maturity, male-specific secondary sex characteristic—AM,can be detected on the 2^(nd) pleopod of males when observed under alaboratory binocular (FIG. 5B, upper panel). This is in contrast to theappendix interna, which is observed in both males and females (FIG. 5B).

In total, 210 females which were given a single injection of suspendedAG cells were grown for a period of 7-8 months under various conditions(earthen ponds, 3.5 cubic meter tanks, etc.). The vast majority of thesemanipulated animals were reared in earthen ponds under grow-outconditions, hence examined only once at the final harvest, 9 months frommanipulation. One hundred females were found to be completely sexreversed into Neo-males, based the developments of AM and two visiblemasculine gonopores at the base of the 5^(th) pereiopods. Additionalfemales presented partial sex reversal with only some of the malecharacteristics observed. Additionally, the three distinctive malemorphotyps in this species (small male, orange claw and blue claw), wereobserved. In a more tightly monitored representative group, 16 females(validated according to a genetic sex marker, FIG. 6) were manipulatedas above. Approximately 50 days post manipulation, all these 16 geneticfemales had developed AM, 13 of which have also developed male gonopores(˜81%) (FIG. 7, center column) thus, considered as Neo-males. Fourteen(14) intact PL males of the same age as the manipulated females, whichhave served as references presented both AM and gonopores at the firstevaluation point (FIG. 6 and FIG. 7 left column), as expected.

The Neo-males were monitored on a weekly basis and found to retain theirAM. Furthermore, Neo-males one year following the manipulationregenerated their AM following a molt regeneration assay. TheseNeo-males were kept in separate tanks, underwent full morphotypicdifferentiation and developed into the dominant male morphotype—the‘blue-claw’ (a representative individual presented in FIG. 8). Withrespect to gonadogenesis, blue-claw Neo-males were dissected and theirreproductive system comprising mature testes (FIG. 9, inset) weresampled for a histological survey (FIG. 9). The dissected individualsrevealed a well-developed sperm duct (FIG. 9A) filled with spermatozoa.Under high magnification the morphology of the cells demonstrated thehallmark structure of the inverted umbrella characterizing this type ofmature cells in M. rosenbergii males (FIG. 9B).

Furthermore, a well-developed testicular tissue comprised of both highlyand lightly stained regions was observed (FIG. 9C). Under highmagnification the highly stained regions, located in the periphery ofany given lobule, was found to accommodate round dividing spermatogoniumcells (FIG. 9D). The lightly stained regions, comprising majority of thelobule's volume were found to accommodate mature spermatozoa.

Example 2 Crossing the Newly Obtained WZ Neo-Males with Natural Females

Mr Neo-males were stocked along with natural females in communal tanks(3.5-10 cubic meters). Once a week, all the females were collected fromthe tank after which, only egg-berried females were removed intoindividual glass tanks. The females were monitored daily until eggs'color had changed from orange to gray by which they were transferred tospawning tanks of 12-15 ppt saline water. After hatching, the femaleswere removed and larvae-culture had commenced as customary. To determinewhether a female was indeed fertilized by a Neo-male, each progeny werekept separately, and immediately after metamorphosis the gender andgenotypic sex of the post larvae were determined as described above.

Females which were stocked along with broodstock Neo-males in communaltanks were successfully fertilized. Upon metamorphosis, PLs weregenetically characterized using the DNA sex markers to verify thatindeed WW PLs were present (FIG. 10) (thus excluding the possibility thefemale was fertilized by a natural ZZ male). In total, out of 865examined individuals representing 4 different crossings of at least 2different Neo-males, 77% (n=668) were positive for the Z chromosome DNAsex marker, namely, WZ females and ZZ males (FIG. 11A). Consequently,the remaining 23% (n=197) were determined as WW females. Furthermore,both the W and Z chromosomes DNA sex markers were used tocomprehensively characterize the precise distribution of genotypes amonga sample of 174 PLs. Of the latter, 42 were identified as ZZ males(24.1%), 90 as WZ females (51.8%) and 42 as WW females (24.1%), with aZZ:WZ:WW genotypic ratio of 1:2.1:1 (FIG. 11B).

Example 3 Crossing WW Females with Natural Males for Obtaining anall-Female WZ Progeny

Intact WW females were grown to sexual maturity and were crossed withnatural ZZ males to obtain 100% all-WZ female (FIG. 12).

Example 4 Crossing on Commercial Scale P Generation

At the parental generation (P), a single broodstock (natural ZZ male andWZ female) produced a progeny comprised of 2,000 to 5,000 of WZsiblings.

F1 Generation

Of the latter, 10 randomly chosen first generation (F1) WZ femalesundergone the sex-reversal manipulation which included celltransplantation as described herein. At least 5 of the chosen WZ femaleswere successfully sex reversed into fully functional WZ Neo-males. Thistrial in which 50% success rate was recorded is in accordance to othersinvolving successful sex-reversal of WZ females into Neo-males.

F2 Generation

Each of these Neo-males (sibling) was grown to sexual maturity and wasallowed to mate once with a natural WZ female. These crossings gave riseto at least five F2 progenies. Each F2 progeny offspring are related ascousins to a parallel F2 offspring of F2. Within each of the mentionedF2 progenies at least 700 WW females were obtained. Obviously, these WWfemales could not have been obtained without the sex-reversalmanipulation of the WZ females and are therefore not a product ofnature.

Each of the F2 WW females obtained from different F2 progenies must becousins produced from crossings involving at least 5 different F1Neo-males (which must be F1 Neo-males brothers) originating from thesame parents. Such cousins were obtained, a minimal number required forproduction.

Example 5 Generation of WW Neo-Male for Obtaining an all-Female WW F3Progeny and the Generation of Various F2 Crossings

Mature male (ZZ) and female (WZ) of the decapod crustacean Macrobrachiumrosenbergii (Mr) were crossed (P generation) to obtain a mixed progenyof post larvae (PL) comprised of 50% males and 50% females. Female PLswere segregated from males by means of molecular genetic sexdetermination using a W-specific DNA sex marker. Prior to PL₁₂₀,identified females were manipulated according to example 1 and grownuntil male-specific secondary sexual characteristics such as developmentof AM and gonopores at the base of the 5^(th) walking legs wereverified.

The F1 females which presented male sexual characteristics were definedas Neo-males and crossed with natural WZ females. This cross gave riseto a F2 progeny with a 3:1 female to male sex ratio. Using theW-specific DNA sex marker, ZZ males were excluded and of the remainingfemales 33% were identified as WW by using a negative screen based on aZ-specific DNA sex marker.

Generation of WW Neo-Males:

Selected WW female PLs from F2 were then manipulated prior to PL₁₂₀according to example 1. Following the manipulation animals were grownuntil secondary sexual characteristics such as developed AM and malegonopores were verified (FIG. 13). Furthermore, development ofmale-specific primary sex characteristics, i.e. gonadogenesis oftesticular tissue, was also validated (FIG. 14). WW females thatpresented male sexual characteristics were defined as WW Neo-males.

The WW Neo-males have undergone full morphotypic differentiation (FIG.15) after which they were crossed with either intact WW females toobtain a 100% all-WW female progeny (FIG. 16; F3 generation) or with WZ(natural) females to obtain a 100% all-female progeny with mixed sexchromosomes composition (F3 generation). Alternatively, intact WWfemales were grown to sexual maturity and are crossed with eithernatural ZZ males to obtain 100% all-WZ female or with WZ Neo-males toobtain a 100% all-female progeny with mixed sex chromosomes composition(F3 generation).

What is claimed is:
 1. A WW homogametic male decapod crustacean.
 2. Amethod for obtaining an all-female progeny, comprising the step ofcrossing a WW female decapod crustacean with another decapod crustacean.3. The method of claim 2, wherein said another WW decapod crustacean isa male.
 4. The method of claim 2, comprising the step of crossing a WWfemale decapod crustacean with a WW male decapod crustacean.
 5. Theprogeny of claim
 2. 6. A female decapod crustacean obtained by themethod of claim
 2. 7. A method for obtaining an all-WW female progeny,comprising the step of crossing a WW male decapod crustacean with a WWfemale decapod crustacean.
 8. A WW female decapod crustacean obtained bythe method of claim
 7. 9. A progeny of a decapod crustacean consistingWW females.
 10. The progeny of claim 9, comprising at least 1,000 fullsisters.
 11. A method for obtaining a WW Neo-male decapod crustacean,comprising the step of injecting to a decapod crustacean comprising theWW sex chromosomes and younger than 180 days post-larva, a compositioncomprising at least 1×10² solitary cells derived from an androgenicgland (AG) of a decapod crustacean, thereby obtaining a WW Neo-maledecapod crustacean.
 12. The method of claim 11, wherein said decapodcrustacean comprising the WW sex chromosomes is a female.
 13. The methodof claim 11, wherein said injecting is injecting into the musculartissue of an abdominal segment.
 14. The method of claim 3, comprisingthe step of crossing a WW female decapod crustacean with a WW maledecapod crustacean.
 15. The progeny of claim
 3. 16. The progeny of claim4.
 17. A female decapod crustacean obtained by the method of claim 3.18. A female decapod crustacean obtained by the method of claim
 4. 19. Afemale decapod crustacean obtained by the method of claim 5.